Explanation Lymphangioleiomyomatosis (LAM), occurring sporadically (S-LAM) or in women with tuberous sclerosis complex (TSC), is a cystic lung disease which appears to result from metastatic dissemination of LAM cells bearing inactivating mutations or having loss of heterozygosity (LOH) in the tumor suppressor genes, TSC1 or TSC2, which leads to hyper-activation of the mammalian target of rapamycin (mTOR). Rapamycin is being used to slow the decline in lung function, to reduce chylous effusions and to shrink the size of AMLs. Thus, the purpose of this study was to determine the effect of rapamycin on circulating LAM cells. Cells from blood obtained by the OncoQuick density-gradient fractionation and from urine and chylous effusions obtained by centrifugation were incubated with anti-CD45-fluorescein isothiocyanate (FITC) and anti-CD235a-R-Phycoerythrin (PE) antibodies, and anti-CD44v6-FITC and anti-CD9-R-PE antibodies, respectively. Cell samples were sorted based on antibody reactivity and LAM cells with TSC2 LOH were identified in 100% of blood specimens and 75% of urine specimens from patients before therapy; in contrast, over a mean duration of 2.2 0.4 (SEM) years of rapamycin therapy, detection rates of LAM cells were significantly decreased to 25% in blood (P < 0.001) and 8% in urine (P = 0.003). Following therapy, greater loss of circulating LAM cells was seen in post-menopausal patients (P = 0.025). Thus, patients receiving rapamycin had a progressive loss of circulating LAM cells, which was dependent on time of treatment and menopausal status. Osteoprotegerin (OPG), a soluble member of the tumor necrosis factor family of receptors, promotes smooth muscle cell proliferation and migration and may act as a survival factor for tumor cells. These cellular mechanisms have been characterized in lymphangioleiomyomatosis (LAM), a multisystem disease affecting primarily women, which is caused by proliferation of smooth muscle-like cells (LAM cells) in lung, lymphatics, and kidneys, resulting in cystic lung destruction, chylous effusions, and renal angiomyolipomas. We hypothesized that OPG may play a role in LAM pathogenesis. Concentrations of OPG were significantly higher in serum from LAM patients than healthy volunteers, and polymorphisms in the promoter of the OPG gene were associated with susceptibility to disease. OPG stimulated proliferation of cells cultured from explanted LAM lungs, and selectively induced migration of LAM cells identified by the loss of heterozygosity for the tumor suppressor gene, Tuberous Sclerosis Complex-2 (TSC2). Cells with TSC2 loss of heterozygosity expressed OPG receptors. LAM lung nodules produced OPG as shown by expression of OPG mRNA, and colocalization of reactivities to anti-OPG and anti-gp100 antibodies in LAM lung nodules. OPG may have tumor promoting roles in the pathogenesis of lymphangioleiomyomatosis, both as an autocrine and systemic factor.